Modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

Abstract

Bacteriophages (phages) subvert their bacterial hosts through the use of phage-encoded proteins called host takeover factors (HTF). HTF target a number of biological processes and their purpose is to either shut off or modulate host biosynthetic machinery. Despite being the most abundant entities on earth, phages and their HTF remain mostly uncharacterised. The Bacillus subtilis infecting phage, SPO1, encodes 26 genes postulated to be HTF of mostly unknown function. These genes are collectively referred to as the host takeover module (HTM). The first objective of the work described in this thesis was to complete the systematic screen of the SPO1 HTM for HTF that attenuate B. subtilis growth. To this end, the effect of expressing the HTF genes in their respective operons was analysed. The HTF with the most pronounced effect were purified and B. subtilis binding partners found. One such HTF was Gp53 which interacts with the AAA+ ATPase ClpC. The major objective of the work described in this thesis was to characterise in detail the SPO1 HTF Gp53. Using bacterial-two hybrid assays, the binding site of Gp53 on ClpC was mapped and mutational analysis identified residues in both proteins required for the interaction. Gp53 was found to stimulate the ATPase activity of ClpC and compete with the native adaptor, MecA, for binding to ClpC. In vitro degradation assays with ClpC and its protease subunit, ClpP, revealed that Gp53 competition alters normal degradation of native substrates. It also appears that, during infection, Gp53 may alter the degradation of SPO1 proteins Gp39 and Gp40. Thus, the results of this study reveal a novel HTF that modulates the activity of the B. subtilis ClpCP protease in an adaptor-like manner and enables efficient SPO1 phage progeny development.